RESUMO
The targeted delivery of genes whose products arrest the cell cycle and/or induce apoptosis represent an important tool for the understanding and controlling forms of unregulated cell growth. The vpr gene product of HIV-1 has been reported to interfere with cell growth and induce apoptosis, but the mechanism of its action is not clearly understood. In order to study these important properties of Vpr, we created a recombinant adenovirus H5.010CMV-vpr (adCMV-vpr) as a tool to deliver the vpr gene to various cell lines to examine its biology. Vpr protein expression was confirmed by Western blot analysis in adCMV-vpr infected cells. We tested the effects of adCMV-vpr on cell growth of several tumor cell lines. Infection of both p53 positive and p53 deficient tumor cell lines with adCMV-vpr resulted in dramatic induction of cell death in short-term assays. We observed that apoptosis was induced through the mitochondrial pathway as we observed changes in the cytochrome c content accompanied by caspase 9 activation. As Bcl-2 is reported to interfere with apoptosis through the mitochondrial pathway, we examined the effect of adCMV-vpr in Bcl-2 over expressing cell lines. We observed that Bcl-2 overexpression does not inhibit adCMV-vpr induced apoptosis. The properties of adCMV-vpr inducing apoptosis through caspase 9 in a p53 pathway independent manner suggest that this is an important reagent. Such a vector may give insight into approaches designed to limit the growth of pathogenic human cells.
Assuntos
Apoptose , Caspases/metabolismo , Produtos do Gene vpr/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Adenoviridae/genética , Apoptose/efeitos dos fármacos , Western Blotting , Caspase 9 , Ciclo Celular , Tamanho Celular , Sobrevivência Celular , Ativação Enzimática , Citometria de Fluxo , Deleção de Genes , Expressão Gênica , Produtos do Gene vpr/genética , Vetores Genéticos/genética , Células HeLa , Humanos , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução Genética , Transgenes/genética , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genéticaRESUMO
OBJECTIVE: To analyze a novel compound, which inhibits serine-threonine protein kinase p38, for its possible bioactivity against HIV-1 infection. METHODS: Proteins involved in cellular signal transduction pathways represent a novel class of host therapeutic targets for infectious diseases. In this regard the serine/threonine kinase p38 MAPK, a member of the mitogen-activated protein (MAP) kinase superfamily of signal transduction molecules may play an important role in HIV-1 infection. We analyzed the ability of this compound (RWJ67657) to inhibit HIV replication in primary T cells and monocytes. Cellular expression of phospho-p38MAPK was studied by Western blot analysis. Blockade of HIV infection induced apoptosis was measured by Annexin V staining. RESULTS: p38 inhibitor RWJ67657 was effective in inhibiting HIV-1 replication in both T-cell and monocyte cell lines, irrespective of the coreceptor used by the virus for entry into the cell. Importantly, both reverse transcriptase and protease resistant escape mutant viruses were effectively suppressed by RWJ67657. In addition, the tested compounds block HIV-induced T-cell apoptosis, a critical means of T-cell depletion linked to AIDS progression. CONCLUSION: Several steps in the HIV-1 virus life cycle appear to depend on cellular activation, including activation of the p38 pathway. Without activation virus replication is thought to be blocked due to incomplete reverse transcription and a lack of proviral DNA integration. The data collectively illustrate that inhibition of the p38 pathway can affect HIV-1 replication. Interruption of HIV infection by p38 inhibitors underscores the value of exploring antiviral drugs that target host cellular proteins.
Assuntos
HIV-1/fisiologia , Imidazóis/farmacologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Piridinas/farmacologia , Linfócitos T/virologia , Replicação Viral/efeitos dos fármacos , Análise de Variância , Anexina A5/análise , Apoptose , Biomarcadores/análise , Western Blotting/métodos , Linhagem Celular , Farmacorresistência Viral , HIV-1/efeitos dos fármacos , Humanos , Células Jurkat , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Monócitos/enzimologia , Monócitos/patologia , Monócitos/virologia , Fosforilação , Linfócitos T/enzimologia , Linfócitos T/patologia , Células U937 , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Human immunodeficiency virus-1 (HIV-1) Vpr encodes a 14 kDa protein that has been implicated in viral pathogenesis through in vitro modulation of several host cell functions. Vpr modulates cellular proliferation, cell differentiation, apoptosis and host cell transcription in a manner that involves the glucocorticoid pathway. To better understand the role of HIV-1 Vpr in host gene expression, approximately 9600 cellular RNA transcripts were assessed for their modulation in primary APC after treatment with a bioactive recombinant Vpr (rVpr) by DNA micro-array. As an extracellular delivered protein, Vpr down-modulated the expression of several immunologically important molecules including CD40, CD80, CD83 and CD86 costimulatory molecules on MDM (monocyte-derived macrophage) and MDDC (monocyte-derived dendritic cells). Maturation of dendritic cells (DC) is known to result in a decreased capacity to produce HIV due to a post-entry block of the HIV-1 replicative cycle. Based on the changes observed in the gene array, we analyzed maturation of DC generated from monocytes in tissue culture as influenced by Vpr. We observed that Vpr-treated immature MDM and MDDC were unable to acquire high levels of costimulatory molecules and failed to develop into mature DC, even in the presence of maturation signals. These studies have importance for understanding the interaction of HIV with the host immune system.
Assuntos
Células Dendríticas/fisiologia , Produtos do Gene vpr/farmacologia , HIV-1/fisiologia , Ativação de Macrófagos/fisiologia , Macrófagos/fisiologia , Fagocitose/fisiologia , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Dendríticas/efeitos dos fármacos , Perfilação da Expressão Gênica , Produtos do Gene vpr/genética , HIV-1/genética , Humanos , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Fagocitose/genética , Vírion/metabolismo , Produtos do Gene vpr do Vírus da Imunodeficiência HumanaRESUMO
The human immunodeficiency virus (HIV) has been reported to target noninfected CD4 and CD8 cells for destruction. This effect is manifested in part through up-regulation of the death receptor Fas ligand (FasL) by HIV-1 negative factor (Nef), leading to bystander damage. However, the signal transduction and transcriptional regulation of this process remains elusive. Here, we provide evidence that p38 mitogen-activated protein kinase (MAPK) is required for this process. Loss-of-function experiments through dominant-negative p38 isoform, p38 siRNA, and chemical inhibitors of p38 activation suggest that p38 is necessary for Nef-induced activator protein-1 (AP-1) activation, as inhibition leads to an attenuation of AP-1-dependent transcription. Furthermore, mutagenesis of the FasL promoter reveals that its AP-1 enhancer element is required for Nef-mediated transcriptional activation. Therefore, a linear pathway for Nef-induced FasL expression that encompasses p38 and AP-1 has been elucidated. Furthermore, chemical inhibition of the p38 pathway attenuates HIV-1-mediated bystander killing of CD8 cells in vitro.
Assuntos
Efeito Espectador , Linfócitos T CD8-Positivos/patologia , Produtos do Gene nef/fisiologia , HIV-1/patogenicidade , Glicoproteínas de Membrana/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Linfócitos T CD8-Positivos/virologia , Morte Celular , Células Cultivadas , Elementos Facilitadores Genéticos , Proteína Ligante Fas , Regulação da Expressão Gênica , Humanos , Fator de Transcrição AP-1 , Ativação Transcricional , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
The HIV-1 accessory protein Vpr exhibits many interesting features related to macrophage and T cell biology. As a viral protein or as a soluble molecule it can suppress immune cell activation and cytokine production in vitro in part by targeted inhibition of NF-kappaB. In this regard we sought to test its effects in vivo on an NF-kappaB-dependent immune pathway. We examined the activity of Vpr in a lethal toxin-mediated challenge model in mice. Intravenous delivery of Vpr was sufficient to protect mice from lethal challenge with staphylococcal endotoxin B (SEB). Furthermore, Vpr protected host CD4+ T cells from in vivo depletion likely by preventing induction of AICD of SEB-exposed cells in a post-toxin-binding fashion. Understanding the biology of Vpr's activities in this model may allow for new insight into potential mechanisms of hyperinflammatory disease and into Vpr pathobiology in the context of HIV infection.
Assuntos
Produtos do Gene vpr/imunologia , HIV-1/imunologia , NF-kappa B/imunologia , Linfócitos T/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Citocinas/imunologia , Endotoxinas/farmacologia , Feminino , Homeostase/imunologia , Técnicas In Vitro , Fígado/imunologia , Fígado/patologia , Ativação Linfocitária , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Staphylococcus , Produtos do Gene vpr do Vírus da Imunodeficiência HumanaRESUMO
HIV-1 sequences are highly diverse due to the inaccuracy of the viral reverse transcriptase. This diversity has been studied and used to categorize HIV isolates into subtypes or clades, which are geographically distinct. To develop effective vaccines against HIV-1, immunogens representing different subtypes may be important for induction of cross-protective immunity, but little data exist describing and comparing the immunogenicity induced by different subtype-based vaccines. This issue is further complicated by poor expression of HIV structural antigens due to rev dependence. One costly approach is to codon optimize each subtype construct to be examined. Interestingly, cis-acting transcriptional elements (CTE) can also by pass rev restriction by a rev independent export pathway. We reasoned that rev+CTE constructs might have advantages for such expression studies. A subtype A envelope sequence from a viral isolate from east Africa was cloned into a eukaryotic expression vector under the control of the CMV-IE promoter. The utility of inclusion of the Mason-Pfizer monkey virus (MPV)-CTE with/without rev for driving envelope expression and immunogenicity was examined. Expression of envelope (gp120) was confirmed by immunoblot analysis and by pseudotype virus infectivity assays. The presence of rev and the CTE together increased envelope expression and viral infection. Furthermore the CTE+rev construct was significantly more immunogenic then CTE alone vector. Isotype analysis and cytokine profiles showed strong Th1 response in plasmid-immunized mice, which also demonstrated the superior nature of the rev+CTE construct. These responses were of similar or greater magnitude to a codon-optimized construct. The resulting cellular immune responses were highly cross-reactive with a HIV-1 envelope subtype B antigen. This study suggests a simple strategy for improving the expression and immunogenicity of HIV subtype-specific envelope antigens as plasmid or vector-borne immunogens.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Anti-HIV/sangue , Proteína gp160 do Envelope de HIV/imunologia , Plasmídeos/genética , Linfócitos T/imunologia , Vacinas de DNA/imunologia , Vacinas contra a AIDS/administração & dosagem , Animais , Citocinas/biossíntese , Testes Imunológicos de Citotoxicidade , Elementos Facilitadores Genéticos/genética , Feminino , Produtos do Gene rev/genética , Proteína gp160 do Envelope de HIV/genética , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1/classificação , HIV-1/genética , HIV-1/imunologia , Humanos , Ativação Linfocitária , Vírus dos Macacos de Mason-Pfizer/genética , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T Citotóxicos , Vacinas de DNA/administração & dosagem , Produtos do Gene rev do Vírus da Imunodeficiência HumanaRESUMO
HIV-1 viral protein R (Vpr) is a virion-associated gene product that profoundly affects T cell proliferation, induces apoptosis and can affect cytokine production in part through interfering with NF-kappa B-mediated transcription from host cells. Collectively, these effects support that Vpr could influence immune activation in vivo. However, this effect of Vpr has not been explored previously. Here we examined the effect of Vpr expression in an in vivo model system on the induction of antigen-specific immune responses using a DNA vaccine model. Vpr co-vaccination significantly altered the immune response to co-delivered antigen. Specifically, in the presence of Vpr, inflammation was markedly reduced compared to antigen alone. Vpr reduced antigen-specific CD8-mediated cytotoxic T lymphocyte activity and suppressed T(h)1 immune responses in vivo as evidenced by lower levels of IFN-gamma. In the presence of Vpr, there is a profound shift in isotype towards a T(h)2 response as determined by the IgG2a:IgG1 ratio. The data support that Vpr compromises antigen-specific immune responses and ultimately effector cell function, thus confirming a strong selective advantage to the virus at the expense of the host.